MDM2 has oncogenic properties when overexpressed and high amounts of the protein are present in human cancers. It is generally accepted that MDM2 promotes tumorigenesis by directly blocking the function of the p53 tumor suppressor. Recent studies indicate that MDM2 induces tumorigenesis by mechanisms that are independent of suppressing p53 function. The pathways in which MDM2 functions to promote cell proliferation and modulate cell cycle progression in a p53-independent manner are not well understood. Dr. Haines and colleagues have recently cloned a novel MDM2 interacting and cell cycle control protein, termed MTBP (for h-IDM2 (Two) binding protein). MTBP functions in a p53-independent manner and its G1 cell cycle arrest activity is blocked by MDM2 overexpression. In addition, they have recently discovered that MDM2 is overexpressed in approximately 20% of primary pancreatic tumors that arise in CDK4R24C/R24C mice. Interestingly, loss of p53 function via p53 gene mutation or pl9ARF gene deletion does not occur in CDK4R24C/R24C-induced tumors that overproduce MDM2 or contain normal amounts of MDM2 protein. Taken together, these results provide evidence for an important role for MDM2 in altering p53 independent pathways controlling cellular proliferation and collaborating with inactivation of pocket proteins during tumorigenesis. To gain further insights into the p53- independent activities of MTBP and MDM2, studies outlined in Project #3 will: 1) define the mechanisms by which MTBP induces G1 cell cycle arrest; 2) determine the mechanism by which MDM2 inhibits MTBP function and if MDM2's transforming capacity is reduced upon eliminating amino acids required for MTBP inactivation; and 3) determine if MDM2 cooperates with mutant CDK4 in cellular transformation via p53-independent mechanisms and if MTBP expression/activity is lost in CDK4R24C/R24C MEFs and tumors.